Bone in vertebrates has both a structural and metabolic function. Its major structural function is to provide support for the body against gravity and to act as a lever system for muscular action. Bone consists of a mineralized phase (essentially calcium hydroxy apatite, approximately 50% by volume) and an organic phase (osteoid) mainly composed of collagen. In addition, bone may be organizationally and functionally subdivided into trabecular (cancellous) and cortical bone. Trabecular bone consists of a three-dimensional gridwork whose individual components (the trabeculae) are plates and struts 100-300 .mu.m thick with the mean intertrabecular space varying between about 500 and 1500 .mu.m. The function of the trabecular structure is to provide the skeleton with mechanical strength. Trabeculation is reduced with a concomitant loss in bone strength as a result of normal aging and disease processes such as osteoporosis.
Aging is associated with a progressive decrease in bone mass that is more prominent in women than in men. For example, most women have lost 40-50% of their peak trabecular mass by the time they are 90 years old while men have lost 10-20% by that age. In addition, symptomatic osteoporosis affects more than 15 million post-menopausal American women. Moreover 25% of all women over the age of 60 years have had at least one spinal compression fracture. The biomechanical and structural aspects of osteoporosis therefore assume exceptional practical importance in clinical medicine. Its diagnosis is similarly of great significance.
Bone mineral density is governed by a variety of metabolic processes controlled by growth hormones, sex hormones and glucocorticoids. For example, estrogen deficiency following menopause or prolonged amenorrhea is known to be associated with bone loss. It is generally recognized that bone mineral density is an important, albeit not complete, descriptor of bone strength. The biomechanical consequences of bone loss during normal aging and osteoporosis have been studied widely See Parfitt, "Age-related Structural Changes in Trabecular and Cortical Bone: Cellular Mechanism and Biomechanical Consequences," Calcif Tissue 1nt 36: $123-5128, 1984; Kleerekoper et al., "The Role of Three-Dimensional Trabecular Microstructure in the Pathogenesis of Vertebral Compression Fractures," Calcif Tissue Int 37: 594-597, 1985; Vesterby et al., "Unbiased Stereological Estimation of Osteoid and Resorption Fractional Surfaces in Trabecular Bone Using Vertical Sections. Sampling Efficiency and Biological Variation," Bone, 1988; Weinstein et al., "Decreased Trabecular Width and Increased Trabecular Spacing Contribute to Bone Loss with Aging," Bone 9: 137-142, 1987.
Two different mechanisms of bone loss have been proposed: Rapid bone loss is believed to be the result of excessive deepening of the osteoclastic resorption cavities, a condition which eventually leads to perforation of the trabeculae, increased size of the marrow spaces and disruption of the bone structure. Slow bone loss results from incomplete refilling by osteoblasts of the resorption cavities. This causes simple thinning of the residual structural elements and thus reduces bone strength in proportion to the amount of bone.
It is also known that bone mass alone is an inadequate descriptor of bone strength. In fact, there is growing evidence that structural parameters may be equally important. Even in studies of large populations there remains considerable overlap between the fracture and normal groups. It has been shown that a quantity of bone mass distributed as widely spaced, disconnected, thick trabeculae is biomechanically less competent than when arranged as numerous, connected, thin plates. In a sex, race and age-matched study of 26 osteoporotic postmenopausal females and 24 control subjects the fractional bone mass was not significantly lower for the subjects with fractures. However, mean trabecular plate density and mean trabecular plate separation were significantly different for the two populations (p &lt;0.001 and p &lt;0.005, respectively (where p represents the probability of the association being fortuitous)). The same group of researchers also found that the loss of trabecular bone that accompanies normal aging results predominantly from a reduction in the number of structural elements with only a slight reduction in the thickness of the remaining elements.
The conventional approach to morphologic measurements of bone makes use of sectioning trabecular bone from frozen specimens, followed by polishing of the surface and subsequent marrow removal. Stereological measurements are then performed on electron micrographs or low magnification (25-30 x) photographs of the cut surfaces of the trabeculae. In this technique the depth-of-field is optimized so that only the cut surfaces of the trabeculae are seen in sharp focus, thus enabling surface measurements to be made without embedding the bone in plastic and sectioning thin slices. This approach is also suitable for image analysis. See Austriaco et al., "Trabecular Bone Densitometry Using Interactive Image Analysis," J Biomed Eng, in press, 1991. Further information about conventional tools and mathematical approaches for stereologic analysis of trabecular bone are available in the literature. See, e.g., Whitehouse, "The Quantitative Morphology of Anisotropic Trabecular Bone," J Microsc 101: 153-168, 1974; Gundersen, et al., "Some New, Simple and Efficient Stereological Methods and Their Use in Pathological Research and Diagnosis," AMPIS 96:379-394, 1988.
Alternative procedures for preparing the bone for stereologic analysis involve embedding the specimen in polymethymethacrylate resin followed by sectioning. The resulting plastic block is machined into thin sections of 100-200 .mu.m thickness. The specimen sections are then further reduced in thickness by polishing to achieve a smooth surface. This procedure is labor intense and requires substantial operator skills. More importantly, however, it only permits measurements in a single plane, thus it may not afford a statistically meaningful measurement of the relevant stereological parameters. Further, by its very nature the method is destructive in that the bone specimen is rendered useless for other measurements, such as analysis in a secondary plane.
Osteoporosis is a widespread disease predominantly afflicting postmenopausal women. As a chronic disease, it may be silent for decades until resulting in fractures late in life. As a result of demineralization and gradual depletion of the trabecular microstructure, the weight-carrying capacity of the bone decreases, leading to atraumatic fractures. The present inventor's copending application Ser. No. 703,411, filed May 21, 1991, provides further background on the causes and effects of osteoporosis and is incorporated by reference herein. The two currently used methods for diagnosis and therapy follow-up are single or dual photon absorptiometry (SPA and DPA, respectively) and quantitative computed tomography (QCT). Those methods, however, are invasive in that they use ionizing radiation. Moreover, their scope is limited in that they measure bone mineral density without providing information on the trabecular microstructure (i.e., the geometry, thickness, orientation and density of the trabecular plates), which is commonly obtained by optical stereology, whereby thin sections of bone biopsy specimens are microscopically analyzed.
Despite extensive research into methods of characterizing bone and the cause and treatment of osteoporosis, there is still a need for noninvasive methods for acquiring information about the trabecular microstructure and for detecting and diagnosing diseases such as osteoporosis that affect trabecular bone.